In nature, many anadromous fish live most of their adulthood in seawater, but swim upstream to freshwater for the purpose of breeding. As a result, anadromous fish hatch from their eggs and are born in freshwater. As these fish grow, they swim downstream and gradually adapt to the seawater.
Fish hatcheries have experienced difficulty in raising these types of fish because the window of time in which the pre-adult fish adapts to seawater (e.g., undergoes smoltification) is short-lived, and can be difficult to pinpoint. As a result, these hatcheries experience significant morbidity and mortality when transferring anadromous fish from freshwater to seawater. Additionally, many of the fish that do survive the transfer from freshwater to seawater are stressed, and consequently, experience decreased feeding, and increased susceptibility to disease. Therefore, these anadromous fish often do not grow well after they are transferred to seawater.
The aquaculture industry loses millions of dollars each year due to problems it encounters in transferring pre-adult anadromous fish from freshwater to seawater. Hence, a need exists to improve methods involved in transferring pre-adult anadromous fish to seawater. A further need exists to increase survival and growth, and reduce stress, of pre-adult anadromous fish that have been transferred to seawater.
The present invention relates to methods for improving the raising of pre-adult anadromous fish or preparing these fish for transfer to seawater by modulating (e.g., increasing and/or decreasing) expression of a receptor, referred to as the Polyvalent Cation Sensing Receptor (PVCR). The modulation of the PVCR includes changes in PVCR protein and mRNA expression as well as changes to the PVCR sensitivity by subjecting the pre-adult anadromous fish to at least one modulator of the PVCR. The pre-adult anadromous fish are subjected to the modulator when it is added to their freshwater environment, and optionally, to the feed. The invention encompasses adding at least one PVCR modulator to the freshwater, and adding feed for fish consumption to the freshwater, wherein the feed has an agent that that is sufficient to contribute to a significantly increased level of the PVCR modulator in serum of the fish. In one embodiment the agent is sodium chloride (NaCl). Hence, the feed contains NaCl and, optionally, at least one PVCR modulator in an amount to contribute to a significantly increased level of a PVCR modulator in the serum of the pre-adult anadromous fish. Modulated expression and/or sensitivity of the PVCR is maintained until the fish are ready to be transferred to seawater. The pre-adult anadromous fish can be maintained in the freshwater having at least one PVCR agonist until they are ready to be transferred to seawater. The present invention also includes optionally exposing the pre-adult anadromous fish to a photoperiod sufficient to increase growth and/or smoltification both before and immediately after their transfer to seawater. Preferably, the photoperiod is continuous. The photoperiod can range between about 12 hours to about 24 hours in a 24 hour period. Additionally, the present invention further includes transferring the fish to seawater where they remain exposed to a continuous photoperiod. The present invention, in one example, allows for these pre-adult anadromous fish weighing as little as about 15 grams or as much as about 120 grams to be transferred to seawater.
In one embodiment of the invention, pre-adult anadromous fish (e.g., salmon, trout and arctic char) are prepared for transfer from freshwater to seawater by adding PVCR agonists, such as calcium and magnesium to the freshwater, and adding feed for fish consumption having between about 1% and about 10% NaCl by weight (e.g. between about 10,000 mg/kg and 100,000 mg/kg) to the freshwater. The amount of calcium added to the freshwater is an amount sufficient to bring the concentration up to between about 2.0 mM and about 10.0 mM, and the amount of magnesium added is an amount sufficient to bring the concentration up to between about 0.5 mM and about 10.0 mM. The feed can optionally include a PVCR agonist, such as an amino acid. A particular amino acid that can be added is tryptophan in an amount between about 1 gm/kg and about 10 gm/kg. The present invention also includes optionally exposing the pre-adult anadromous fish to a photoperiod for a sufficient amount of time to increase growth and/or smoltification. Preferably, the photoperiod is continuous (e.g., for a period of between about 12 hours and about 24 hours in a 24 hour period). A continuous photoperiod can occur for 1 day to several days, as described herein, before and after seawater transfer.
Additional embodiments of the invention include methods of increasing or improving food consumption before and/or after seawater transfer, increasing growth, increasing survival and/or reducing mortality, improving the Feed Conversion Ratio (FCR), increasing the Specific Growth Rates (SGR), reducing osmotic damage, transferring parr (e.g., between about 15 and about 60 grams) to seawater, and transferring pre-adult anadromous fish to seawater having an elevated temperature of about 14xc2x0 C. to about 19xc2x0 C. These methods are performed by adding at least one PVCR modulator to the freshwater, subjecting or exposing the pre-adult anadromous fish to at least one PVCR modulator, or introducing the pre-adult anadromous fish to freshwater having at least one PVCR modulator, in an amount sufficient to modulate expression and/or sensitivity of the PVCR. The methods also involve adding feed having between about 1% and about 10% NaCl by weight to the freshwater and transferring the pre-adult anadromous fish to seawater.
The present invention also embodies methods for increasing Sodium Potassium ATPase (Na+K+ATPase) activity in chloride cells in tissues (e.g., gill) of pre-adult anadromous fish or reducing the frequency of chloride cells in secondary lamellae of gill of pre-adult anadromous fish. The method comprises adding a PVCR modulator to the freshwater in an amount sufficient to modulate expression and/or sensitivity of at least one PVCR; and adding feed for fish consumption to the freshwater, wherein the feed contains an amount of NaCl sufficient to contribute to a significantly increased level of the PVCR modulator in serum of the pre-adult anadromous fish. An increase in Na+ K+ATPase activity in chloride cells and/or a reduction in the number of chloride cells in secondary lamellae occurs, as compared to fish of the same size and age group held in freshwater and not subjected to steps of the present invention. A ratio of the distribution of chloride cells between secondary lamellae (SL) and primary lamellae (PL) can be determined. The SL/PL ratio decreases, as compared to fish maintained in freshwater and not subjected to steps of the present invention. The methods of the present invention reduce the SL/PL ratio to closely resemble the ratio exhibited by fish already transferred to seawater, yet the treated fish are still maintained in freshwater. The SL/PL ratio can range between about 0.1 and about 1.0.
In other embodiments, the invention encompasses detection assays or methods of determining whether pre-adult anadromous fish that are subjected to at least one PVCR modulator and are fed with feed having between about 1% and about 10% NaCl by weight, are ready for transfer to seawater, by assessing the amount and/or localization of PVCR expression in the pre-adult anadromous fish. A modulated (e.g., increased or decreased) level of expression and/or sensitivity, as compared to a control (e.g., PVCR expression from a fish not subjected to a PVCR modulator), indicates that the pre-adult anadromous fish are ready for transfer to seawater. In a preferred embodiment, the assay includes contacting an anti-PVCR antibody with a sample (e.g., gill, skin, intestine, olfactory lamellae, urinary bladder, kidney, brain or muscle) under conditions sufficient for the formation of a complex between the antibody and the PVCR; and detecting the formation of the complex. In another embodiment the assay relates to hybridizing a nucleic acid sequence having a detectable label to the nucleic acid sequence of the PVCR of a sample taken from the pre-adult anadromous fish and detecting the hybridization. In yet another embodiment, detection of the PVCR in a tissue can also be accomplished by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). This embodiment involves reverse transcribing mRNA from the tissue having at least one PVCR; performing a PCR reaction with PVCR-specific primers to obtain RT-PCR product; and determining the presence or amount of the PVCR.
In yet another embodiment, the present invention relates to various compositions and mixtures. In particular, the invention pertains to an aquatic food composition having a concentration of NaCl between about 10,000 mg/kg and 100,000 mg/kg (e.g., about 12,000 mg/kg). The aquatic food composition can optionally include a PVCR modulator (e.g. tryptophan in an amount between 1 gm/kg and 10 gm/kg).
The invention also embodies an aquatic mixture for providing an environment to improve the raising of pre-adult anadromous fish. The mixture includes at least one PVCR modulator. An example of such a mixture is a calcium source, that when added to freshwater, provides a concentration of between about 2.0 mM and about 10.0 mM; and a magnesium source, that when added to freshwater, provides a concentration of between about 0.5 mM and 10.0 mM.
In yet another embodiment, the present invention relates to kits. In particular, the invention embodies kits for improving the raising of pre-adult anadromous fish, that includes a PVCR modulator for addition to the freshwater and an aquatic food composition, as described herein. In another embodiment, the invention includes kits for determining whether a pre-adult anadromous fish are ready for transfer to seawater, after being subjected to at least one PVCR modulator and feed having between about 1% and about 10% NaCl by weight. The kit includes either an anti-PVCR antibody, and a solid support; or a nucleic acid sequence having a detectable label that can hybridize to nucleic acid of an aquatic PVCR.
Surprisingly, it has been discovered that modulated expression and/or altering the sensitivity of the PVCR allows these pre-adult anadromous fish to better adapt to seawater. Until the discovery of the present invention, the aquaculture industry was unable to transfer the pre-adult anadromous fish to seawater without subjecting the fish to stress, death and/or disease. Unlike this practice, carrying out the steps of the invention modulates the expression and/or alters the sensitivity of the PVCR and allows for transfer of the pre-adult anadromous fish to seawater with minimal or no stress, death and/or disease, and unexpectedly provides several benefits, such as increased growth and the ability to transfer these fish to water having higher temperatures, as further described herein. The present invention results in one or more of the following advantages in transferring pre-adult anadromous fish to seawater: a reduction in mortality; improvement in feeding; an increase in growth; a decrease in the amount of diseased fish; and/or a reduction in osmotic shock. The present invention also allows for earlier harvesting of the fish with increased flexibility in producing fish year round. Additionally, the methods of the present invention can result in significant cost savings for fish hatcheries.